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Natriuretic fusion proteins

a fusion protein and natriuretic technology, applied in the field of fusion proteins with diuretic, natriuretic, vasodilatory activity, can solve the problems of short half-life of proteins, limited previous therapeutic administration of peptides, and inability to achieve the effect of preventing bacterial infection

Inactive Publication Date: 2010-12-09
BOEHRINGER INGELHEIM INT GMBH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]In another aspect, the invention relates to methods to treat or ameliorate pathological conditions in which activation of the NPRA receptor confers a therapeutic benefit on the patient, including, but not limited to, diseases associated with abnormal diruretic, natriuretic and vasodilatory activity and / or in which it is desirable to induce naturesis, diuresis, vasodilation or to modulate the renin-angiotensin II and aldosterone systems. These conditions include those that may be characterized by an excess in extraceullar fluid, including, but not limited to, pulmonary edema. In a particularly preferred embodiment, the invention includes methods to treat or ameliorate pathological conditions of the cardiovascular system including, but not limited to, chronic heart failure (non-ischemic), post-MI heart failure (ischemic CHF), acute MI, reperfusion injury, left ventricular dysfunction (LVD), cardiac fibrosis, diastolic heart failure, and hypertrophic cardiomyopathy. In addition, hypertensive disorders including, but not limited to hypertension, e.g., pulmonary hypertension, systolic hypertension, resistant hypertension and other cardiovascular related diseases such as diabetic nephropathy may be treated or ameliorated by the methods of the present invention. It is also contemplated herein that the fusion proteins and pharmaceutical compositions of the present invention may provide therapeutic benefit for patients undergoing coronary artery bypass graft surgery (CABG).

Problems solved by technology

Unfortunately, despite this promising clinical application, the therapeutic usefulness of ANP and BNP is sorely limited as endopeptidase degradation, as well as natriuretic peptide clearance receptor (NPR-C) mediated internalization, causes these proteins to have a fairly short half-life in vivo.
Thus, previous therapeutic administration of these peptides has been limited to time consuming intravenous infusion, typically in a hospital or other medical care facility.

Method used

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Examples

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example 1

Structural Modeling to Predict Minimum Linker Distance

[0198]Structural modeling was done to determine the linker requirements for the Fc and ANP fusions. With the both molecules oriented to minimize distance as well as minimize steric and electrostatic repulsions, a minimal distance from the C-terminus of ANP is 12 Å from the closest N-terminus of the Fc dimer and 17 Å from the other N-terminus of the Fc dimer. If the Fc dimer has only one ANP fused, a 4 to 6 aa minimum linker length would be suggested. With two ANP peptides bound to the Fc dimer, if only one ANP bound (in a 1:1 Fc dimer:NPRA ratio) then a minimum linker length of 9 aa for each linker would be suggested. For both ANP's to bind in a 1:2 Fc dimer:NPRA ratio, linkers with a minimum length of 12 aa would be suggested.

example 2

Synthesis and Characterization of Synthetic ANP Fusion Proteins

[0199]Synthetic chemistry was used to generate hANP28 peptides with linkers fused to human Fc of IgG1 isotype. The resulting semi-synthetic ANP-Fc fusion molecules generated are shown in Table 2. The linkers tested were glycine succinate (L1), GlyGly (L2), (GlyGlySer)3GlyGly (L4) (SEQ ID NO: 3) and Gly(SerGlyGly)2SerGly (L3) (SEQ ID NO: 2). The glycines (G) were used to add flexibility, while the polar serines (S) were built in to add solubility. The hANP28 peptides with linkers were linked to recombinantly produced Cys-Fc in two orientations: 1) C-terminus of hANP28+Linker fused to N-terminus of Cys-Fc [Orientation #1], and 2) N-terminus of Linker+hANP28 fused to N-terminus of Cys-Fc [Orientation #2]. The synthetic chemistry of the first orientation resulted in a complete peptide bonded structure while the chemistry of the second orientation left a succinate moiety in place of one amino acid of the fusion.

[0200]The semi...

example 3

Recombinant ANP Fusion Proteins

[0205]A production platform for the rapid generation of recombinant ANP-Fc fusion proteins was generated. The process starts with “base” Fc fusion vectors that allows DNA cassettes to be rapidly and seamlessly inserted onto the N-terminus of either IgG1 or IgG2 Fc. The Fc fusions to both IgG1 and IgG2 isotypes were generated in such a way that the hinge region is chopped down to the same CPPCP hinge residues thus ensuring that the linker extension and ANP fusion would be equally extended on the two isotypes. The respective DNA and protein sequences of the four recombinant ANP-Fc fusion proteins generated are represented by SEQ ID NOs: 24-31 (see, e.g., FIG. 1A-B). These fusion proteins each comprise a N-terminal mouse IgG kappa light chain signal sequence METDTLLLWVPGSTG (SEQ ID NO: 32) that is cleaved off and not part of the final protein product. The bolded The ANP-Fc fusion constructs were initially produced using 1 L transient mammalian expression ...

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Abstract

Natriuretic peptide fusion proteins comprising natriuretic peptides linked to antibody Fc domains, nucleic acid molecules encoding the fusion proteins disclosed herein, expression vectors expressing said fusion proteins, pharmaceutical compositions comprising said fusion proteins, and methods for their therapeutic use are disclosed.

Description

STATEMENT REGARDING JOINT RESEARCH AGREEMENT[0001]One or more inventions contained in this application were developed under a joint research agreement as defined in the Cooperative Research and Technology Enhancement Act of 2004 between Boehringer Ingelheim International, GmbH and Syntonix Pharmaceuticals, Inc.FIELD OF THE INVENTION[0002]This invention relates to fusion proteins having diuretic, natriuretic, and vasodilatory activity, specifically natriuretic peptides linked to antibody Fc domains. The fusion proteins of this invention exhibit extended plasma half-life compared to wild type natriuretic peptides, which serve as ligands to membrane guanylyl cyclases. The invention is also directed to nucleic acid molecules encoding the fusion proteins disclosed herein, expression vectors expressing said proteins, pharmaceutical compositions comprising the fusion proteins and / or nucleic acid molecules of the present invention. Compositions according to this invention may be administere...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/00C07K14/00C12N15/12C12N15/63C12N5/00C12P21/00A61P9/00A61P31/04A61P11/00A61P13/12
CPCA61K38/00C12N15/62C07K14/58
Inventor CANADA, KEITHSCHOENBECK, UWEHICKEY, EUGENE R.MEZO, ADAMMCDONNELL, KEVIN
Owner BOEHRINGER INGELHEIM INT GMBH
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